Human Lung Innate Immune Response toBacillus anthracisSpore Infection

Abstract

Bacillus anthracis, the causative agent of inhalational anthrax, enters a host through the pulmonary system before dissemination. We have previously shown that human alveolar macrophages participate in the initial innate immune response to B. anthracis spores through cell signal-mediated cytokine release. We proposed that the lung epithelia also participate in the innate immune response to this pathogen, and we have developed a human lung slice model to study this process. Exposure of our model to B. anthracis (Sterne) spores rapidly activated the mitogen-activated protein kinase signaling pathways ERK, p38, and JNK. In addition, an RNase protection assay showed induction of mRNA of several cytokines and chemokines. This finding was reflected at the translational level by protein peak increases of 3-, 25-, 9-, 34-, and 5-fold for interleukin-6 (IL-6), tumor necrosis factor alpha, IL-8, macrophage inflammatory protein 1␣/␤, and monocyte chemoattractant protein 1, respectively, as determined by an enzyme-linked immunosorbent assay. Inhibition of individual pathways by UO126, SP600125, and SB0203580 decreased induction of chemokines and cytokines by spores, but this depended on the pathways inhibited and the cytokines and chemokines induced. Combining all three inhibitors reduced induction of all cytokines and chemokines tested to background levels. An immunohistochemistry analysis of IL-6 and IL-8 revealed that alveolar epithelial cells and macrophages and a few interstitial cells are the source of the cytokines and chemokines. Taken together, these data showed the activation of the pulmonary epithelium in response to B. anthracis spore exposure. Thus, the lung epithelia actively participate in the innate immune response to B. anthracis infection through cell signal-mediated elaboration of cytokines and chemokines.

Nonhematopoietic Cells Are Key Players in Innate Control of Bacterial Airway Infection

“…Furthermore, nonhematopoietic cells in the lung were shown to contribute to cytokine production upon exposure to Bacillus anthracis spores (46) or Haemophilus influenzae infection (47). Finally, in P. aeruginosa-infected mouse lungs, MyD88 in nonhematopoietic cells is essential for early control of infection, whereas at later phases of the infection, MyD88 expression in both parenchymal and hematopoietic cells contributes to control pathogen replication (48).…”

TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation

“…Several studies have investigated cytokine secretion from sliced human lung tissue in response to various stimuli (3,4), however, the tissue was not necessarily selected for the presence of an airway. Hence, we adapted an integrated model system that allows for the assumption that all structural components of the lung, including ASM and epithelial cells as well as parenchymal tissue, are present and intact.…”

Cigarette smoke extract suppresses the RIG-I-initiated innate immune response to influenza virus in the human lung

“…Lung slices were exposed to 6 ϫ 10 6 PFU/ml of influenza virus or virus diluents with or without 2% CSE treatment. Brefeldin A (LC Laboratories, Wofford, MA) was added at a concentration of 5 g/ml to block protein export to enhance cytokine detection (9). After the incubation, lung slices were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min and then imbedded in paraffin.…”

“…Only tissue that did not contain tumor was used for experiments. The tumor-free lung tissue was transported in sterile PBS (pH 7.2) containing 200 g gentamicin/ml, 100 U penicillin/ml, 100 g streptomycin/ml, and 2.5 g amphotericin B/ml (PBS ϩ antibiotics), and the tissue was subsequently stored at 4°C in PBS ϩ antibiotics for no longer than 4 h. Lung segments were inflated with lung slice medium (LSM) containing 1.5% agarose, 1-cm cores were prepared, and cores were sliced into 500-m-thick sections as previously described (9). LSM consisted of minimal essential medium (Sigma, St. Louis, MO) supplemented with 1.0 g bovine insulin/ml, 0.1 g hydrocortisone/ml, 0.1 g retinyl acetate/ ml, 200 g gentamicin/ml, 100 U penicillin/ml, 100 g streptomycin/ ml, and 1.25 g amphotericin B/ml.…”

“…We (6,9) have developed a human lung organ culture model to study the local lung response to human pathogens. Precision-cut lung slices have been used in toxicology studies and have advantages over the use of isolated, cultured epithelial cells for the study of infectious disease.…”